Spirobenzofuran lactams and their derivatives, processes for their preparation and use thereof

ABSTRACT

The present invention relates to spirobenzofuran lactam derivatives of the formula I 
                         
which are formed during fermentation by the microorganism  Stachybotris atra  ST002348, DSM 14952, processes for their preparation, their use as pharmaceuticals, and the microorganism  Stachybotris atra  ST002348, DSM 14952.

This application claims the benefit of U.S. Provisional Application No.60/465,999, filed Apr. 28, 2003, and incorporated herein by reference.

The present invention relates to novel active compounds (spirobenzofuranlactam derivatives), which are formed during fermentation by themicroorganism Stachybotris atra ST002348, DSM 14952, processes for theirpreparation, their use as pharmaceuticals, pharmaceuticals containingspirobenzofuran lactam derivatives and the microorganism Stachybotrisatra ST002348, DSM 14952.

The plasminogen activation system comprises an enzyme cascade whichmakes possible the controlled, localized formation of the proteolyticenzyme plasmin. The formation of plasmin plays an important role in amultiplicity of physiological and pathophysiological processes. Withinthis enzyme cascade, the conversion of the zymogen plasminogen into theproteolytically active plasmin is activated either by tPA (tissue typeplasminogen activator) or uPA (urokinase type plasminogen activator).The plasmin activity can be controlled or regulated at different levels.The activity of tPA and uPA is in turn controlled by PAI-1 and PAI-2.While tPA is the most important plasminogen activator in fibrinolysis,uPA plays an important role in plasmin formation at sites where adegradation of extracellular matrix takes place. While uPA is regulatedboth by PAI-1 (plasminogen activator inhibitor 1) and PAI-2, there areindications that tPA is influenced only by PAI-1. PAI-1 thus plays animportant role in the maintenance of the equilibrium between fibrinformation and fibrinolysis (J. D. Vassalli et al., J. Clin. Invest.,1991, 88, 1067–1072). Many studies yielded information that an increasedPAI-1 level is a risk factor for cardiovascular diseases. Elevated PAI-1concentrations were detected, inter alia, in the case of coronary heartdisease, acute myocardial infarct, unstable angina pectoris, venousthrombosis and venous thromboembolisms (P. J. Declerck et al., J.Intern. Med., 1994, 236, 425–432; H. A. Dawsons, Atherosclerosis, 1992,95, 105–117). These clinical studies point to the fact that PAI-1 is anovel target for the treatment of diseases which accompany decreasedfibrinolysis, for example decreased wound healing (Charlton P., Exp.Opin. Invest. Drugs, 1997, 6, 539–554).

Elevated PAI-1 values are also connected with arterial thrombosis,arteriosclerosis, insulin resistance and macrovascular injuries in typeII diabetes mellitus patients, hypoxia, septic shock, pneumonia andpulmonary fibrosis, and moreover with cancers, in particular breastcancer, intestinal cancer, gastric cancer, hepatic cancer, brain tumors,ovarian tumors, esophageal cancer, renal cancer, muscle cell carcinoma,in particular head and neck muscle carcinoma, PAI-1 being credited witha key role in the progress and the metastasis of cancers, in particularin the proteolysis, adhesion, mobilization, invasion, chemotaxis,proliferation and angiogenesis (P. Carlton, Exp. Opin. Invest. Drugs,(1997), 6(5), 539–554; F. Frankenne et al., Expert Opinion onTherapeutic Targets, 1999, 3(3), 469–481). A treatment of the diseasesmentioned by way of introduction is therefore possible by inhibition ofPAI-1.

It is therefore the object of the present invention to make availableinhibitors of the plasminogen activator inhibitor 1 (PAI-1).

It has surprisingly being found that the microorganism strainStachybotris atra ST002348, DSM 14952, is able to form compounds whicheffectively inhibit the plasminogen activator inhibitor 1 (PAI-1) invery low concentrations. The compounds of the formula (I) inhibit theinhibition of the enzymatic activity of tPA by PAI-1 and are accordinglysuitable for the treatment and/or prophylaxis of coronary heart disease,acute myocardial infarct, unstable angina pectoris, venous thrombosisand venous thromboembolisms, arterial thrombosis, arteriosclerosis,insulin resistance and macrovascular injuries in type II diabetesmellitus patients, hypoxia, septic shock, pneumonia and pulmonaryfibrosis, and cancers, in particular breast cancer, intestinal cancer,gastric cancer, hepatic cancer, brain tumors, ovarian tumors, esophagealcancer, renal cancer, muscle cell carcinoma, in particular head and neckmuscle carcinoma. In particular, the compounds according to theinvention are suitable for an antithrombotic therapy for treatment andprophylaxis in patients with coronary heart diseases and antithromboticdiseases of the peripheral venous system.

The invention thus relates to compounds of the formula (I), also calledspirobenzofuran lactam derivatives below,

where;

-   R¹ and R² independently of one another are H, C₁–C₆-alkyl,    C₂–C₆-alkenyl, C₂–C₆-alkynyl or C₅–C₁₄-aryl, in which alkyl,    alkenyl, alkynyl and aryl are unsubstituted or mono- to    trisubstituted by a radical from the group consisting of —OH, ═O,    —O—C₁–C₆-alkyl, —O—C₂–C₆-alkenyl, —O—C₅–C₁₄-aryl, —NH—C₁–C₆-alkyl,    —NH—C₂–C₆-alkenyl, —NH[—C(═O)—(C₁–C₆-alkyl)],    —NH[—C(═O)—(C₅–C₁₄-aryl)], —NH₂ or halogen,-   R³ is —OH, —O—R¹ or —NH—R¹, and-   R⁴ is H, C₁–C₆-alkyl, C₂–C₆-alkenyl, C₅–C₁₄-aryl or    —(C₁–C₆-alkyl)-(C₅–C₁₄-aryl),-   and their physiologically tolerable salts and/or obvious chemical    equivalents.

Preferably, R¹ and R² independently of one another are H or C₁–C₆-alkyl,particularly preferably H, R³ is OH or —O—C₁–C₆-alkyl, particularlypreferably OH, and R⁴ is C₁–C₆-alkyl or —(C₁–C₆-alkyl)-(C₅–C₁₄-aryl),particularly preferably benzyl, 2-butyl or 1-(2-methylpropyl).

A compound of the formula (I) is particularly preferred where R¹ and R²are H, R³ is OH and R⁴ has the abovementioned general or preferredmeaning.

The invention further preferably relates to a compound of the formula(I) characterized by a compound of the formula (II),

a compound of the formula (III) or

a compound of the formula (IV)

or a physiologically tolerable salt thereof.

Chiral centers in the compounds of the formula (I), (II), (III) and (IV)can, unless stated otherwise, be present in the R or in the Sconfiguration. The invention relates both to the optically purecompounds and stereoisomer mixtures such as enantiomer mixtures anddiastereomer mixtures.

C₁–C₆-alkyl is a straight-chain or branched alkyl having 1 to 6 C atoms,preferably having 1 to 4 C atoms, e.g. methyl, ethyl, i-propyl,tert-butyl and hexyl.

C₂–C₆-alkenyl is a straight-chain or branched alkenyl having 2 to 6 Catoms, which is mono-, di- or triunsaturated, e.g. allyl, crotyl,1-propenyl, penta-1,3-dienyl and pentenyl.

C₂–C₆-alkynyl is a straight-chain or branched alkynyl having 2 to 6 Catoms, which is mono- or diunsaturated, e.g. propynyl, butynyl andpentynyl.

C₅–C₁₄-aryl is an aryl group having 5 to 14 C atoms, e.g. phenyl, benzylor 1- or 2-naphthyl, which are substituted or unsubstituted by one, twoor three substituents from the group consisting of halogen, e.g.chlorine, bromine, fluorine, C₁–C₄-alkyl, e.g. methyl, hydroxyl,C₁–C₄-alkoxy, e.g. methoxy or by trifluoromethyl.

Halogen is an element from the group consisting of fluorine, chlorine,bromine and iodine.

The invention further relates to a process for the preparation of acompound of the formula (I), which comprises fermenting the strainStachybotris atra ST002348, DSM 14952, or one of its variants or mutantsunder suitable conditions in a culture medium until one or more of thecompounds of the formula (I) accumulate in the culture medium and thenisolating it from the culture medium and optionally converting it intochemical equivalents and/or a physiologically tolerable salt.

Preferably, the strain Stachybotris atra ST002348, DSM 14952, itsmutants and/or variants is fermented in a nutrient solution or a solidmedium (also called culture medium) having a carbon and nitrogen sourceand the customary inorganic salts until the compounds according to theinvention accumulate in the culture medium, then the compounds areisolated from the culture medium and optionally separated into theindividual active components.

The process according to the invention can be employed for fermentationon the laboratory scale (milliliter to liter range) and for theindustrial scale (cubic meter scale).

A heavily producing colony of Stachybotris atra was proliferated in apreculture. The strain was deposited in the Deutsche Sammlung vonMicroorganismen und Zellkulturen GmbH, Mascheroder Weg 1B, 3300Brunswick, Germany, according to the rules of the Budapest convention onApr. 23, 2002 under the number DSM 14952 or the reference ST002348 ofAventis Pharma Deutschland GmbH allocated by the depositor.

On malt agar, Stachybotris atra ST002348, DSM 14952, has a white toorange-colored mycelium. The strain forms hyaline ellipsoid tubercularconidia (8–11×6–11 μm) characteristic of the species.

Instead of the strain Stachybotris atra ST002348, DSM 14952, it is alsopossible to employ its mutants and variants which synthesize one or moreof the compounds according to the invention.

A mutant is a microorganism in which one or more genes of the genomehave been modified, the gene or genes which are responsible for theability of the organism to produce the inventive compound being retainedfunctionally and hereditarily.

Such mutants can be produced in a manner known per se by physical means,for example irradiation, such as with ultraviolet rays or X-rays, orchemical mutagens, such as, for example, ethyl methanesulfonate (EMS);2-hydroxy-4-methoxybenzophenone (MOB) orN-methyl-N′-nitro-N-nitroso-guanidine (MMNG), or as described by Brocket al. in “Biology of Microorganisms”, Prentice-Hall, pages 238–247(1984).

A variant is a phenotype of the microorganism. Microorganisms have theability to adapt to their environment and therefore show markedphysiological flexibility. In the case of phenotypic adaptation, allcells of the microorganism are involved, the nature of the alterationnot being genetically conditioned and being reversible under alteredconditions (H. Stolp, Microbial ecology: organisms, habitats,activities. Cambridge University Press, Cambridge, GB, page 180, 1988).

The screening for mutants and variants which synthesize one or more ofthe compounds according to the invention is carried out according to thefollowing scheme:

-   -   preparation of mutants and/or variants according to methods        known per se;    -   culturing of the mutants and/or variants obtained in this way;    -   lyophilization of the shaker cultures;    -   extraction of the lyophilizates using an organic solvent;    -   extraction of the compound from the culture filtrate using solid        phases;    -   analysis by means of HPLC, TLC or by testing the biological        activity;    -   optionally elucidation of the taxonomy of the mutants and/or        variants.

The fermentation conditions described below apply for Stachybotris atraST002348, DSM 14952, and mutants and variants thereof.

In a nutrient solution which contains a carbon source and a nitrogensource and the customary inorganic salts, Stachybotris atra ST002348,DSM 14952 produces the spirobenzofuran lactam derivatives.

Suitable preferred carbon sources for the aerobic fermentation areassimilable carbohydrates and sugar alcohols, such as glucose, lactose,sucrose or D-mannitol, and carbohydrate-containing natural products,such as, for example, malt extract or starch. Possiblenitrogen-containing nutrients are: amino acids; peptides; proteins;degradation products of proteins and peptides, in particular peptidesobtained synthetically or biosynthetically, for example casein, peptonesor tryptones; meat extracts; yeast extracts; ground seeds, for exampleof corn, wheat, beans, soybeans or the cotton plant; distillationresidues of alcohol production; meat meals or yeast extracts; ammoniumsalts; nitrates. Customary inorganic salts are, for example, chlorides,carbonates, sulfates or phosphates of an alkali metal or alkaline earthmetal, of iron, zinc, cobalt or manganese.

The formation of the compounds according to the invention proceedsparticularly readily in a nutrient solution which contains approximately0.1 to 5%, preferably 0.5 to 2%, of starch, 0.2 to 5%, preferably 0.5 to1%, of yeast extract and 0.2 to 5%, preferably 0.5 to 2%, of glucose.The data in percent are in each case based on the weight of the totalnutrient solution.

In this nutrient solution, Stachybotris atra ST002348, DSM 14952, formsa mixture of spirobenzofuran lactam derivatives. Depending on thecomposition of the nutrient solution, the quantitative proportion of oneor more of the spirobenzofuran lactam derivatives according to theinvention can vary.

The microorganism is cultured aerobically, i.e., for example, submersedwith shaking or stirring in shaker flasks or fermenters, optionally withintroduction of air or oxygen. It can be carried out in a temperaturerange from approximately 18 to 35° C., preferably at approximately 20 to30° C., in particular at 22 to 27° C. The pH range should be between 4and 8, preferably between 5 and 6. The microorganism is in generalcultured under these conditions for a period of 48 to 200 hours,preferably 72 to 168 hours.

Advantageously, the culturing is carried out in a number of stages, i.e.first one or more precultures are prepared in a liquid nutrient medium,and are then inoculated into the actual production medium, the mainculture, for example in the volume ratio 1:10 to 1:100. The precultureis obtained, for example, by inoculating a mycelium into a nutrientsolution and allowing it to grow for approximately 36 to 120 hours,preferably 48 to 72 hours. The mycelium can be obtained, for example, byallowing the strain to grow for approximately 3 to 21 days, preferably 4to 10 days, on a solid or liquid nutrient medium, for example malt-yeastagar or potato-dextrose agar.

The course of the fermentation can be monitored by means of the pH ofthe cultures or of the mycelium volume and by chromatographic methods,such as, for example, high-performance liquid chromatography (HPLC), ortesting the biological activity.

The isolation process described below serves for the purification of thespirobenzofuran lactam derivatives of the formula (I) according to theinvention:

The isolation or purification of a spirobenzofuran lactam derivativeaccording to the invention from the culture medium is carried outaccording to known methods taking into account the chemical, physicaland biological properties of the natural substances. To test theconcentration of the respective spirobenzofuran lactam derivatives inthe culture medium or in the individual isolation stages, HPLC can beused, the amount of the substance formed expediently being compared witha calibration solution.

For isolation, the culture broth or the culture together with the solidmedium are lyophilized, then the spirobenzofuran lactam derivatives areextracted from the lyophilizate using an organic solvent which isoptionally miscible with water. The organic solvent phase contains thenatural substances according to the invention; it is optionallyconcentrated in vacuo and further purified.

The further purification of one or more compounds according to theinvention is carried out by chromatography on suitable materials,preferably, for example, on molecular sieves, on silica gel, alumina, onion exchangers or on adsorber resins or on reversed phases (RP). Withthe aid of this chromatography, the spirobenzofuran lactam derivativesare separated. The chromatography of the spirobenzofuran lactamderivatives is carried out using buffered aqueous solutions or mixturesof aqueous and organic solutions.

Mixtures of aqueous or organic solutions are understood as meaning allorganic solvents miscible with water, preferably methanol, 2-propanoland acetonitrile, in a concentration of 5 to 80% of solvent, preferably20 to 50% of solvent or else any buffered aqueous solutions which aremiscible with the organic solvents. The buffers to be used are the sameas indicated above.

The separation of the spirobenzofuran lactam derivatives on the basis oftheir differing polarity is carried out with the aid of reversed phasechromatography, for example on MCI® (adsorber resin from Mitsubishi,Japan) or Amberlite XAD® (TOSOHAAS), or on further hydrophobicmaterials, such as, for example, on RP-8 or RP-18 phases. Moreover, theseparation can be carried out with the aid of normal phasechromatography, for example on silica gel, alumina and the like.

The chromatography of the spirobenzofuran lactam derivatives is carriedout using buffered or acidified aqueous solutions or mixtures of aqueoussolutions with alcohols or other water-miscible organic solvents. Theorganic solvent used is preferably 2-propanol and acetonitrile.

Buffered or acidified aqueous solutions are understood as meaning, forexample, water, phosphate buffer, ammonium acetate, citrate buffer in aconcentration of up to 0.5 M and formic acid, acetic acid,trifluoroacetic acid or all commercially available acids known to theperson skilled in the art, preferably in a concentration of up to 1%. Inthe case of buffered aqueous solutions, 0.1% ammonium acetate isparticularly preferred.

Chromatography was carried out using a gradient which begins with 100%water and ends with 100% solvent; a linear gradient of 20 to 100% of2-propanol or acetonitrile was preferably used.

Alternatively, gel chromatography or chromatography on hydrophobicphases can also be carried out. Gel chromatography is carried out onpolyacrylamide or copolymer gels, such as, for example, Biogel-P 2®(Biorad) or Fractogel TSK HW 40® (Merck, Germany or Toso Haas, USA). Thesequence of the aforementioned chromatographies is reversible.

The present invention furthermore relates to all obvious chemicalequivalents of the compounds of the formula (I) according to theinvention. Such equivalents are compounds which exhibit a slightchemical difference, i.e. have the same action or are converted into thecompounds according to the invention under mild conditions. Theequivalents mentioned also include, for example, salts, reductionproducts, oxidation products, esters, ethers, acetals or amides of thecompounds of the formula (I) and equivalents which the person skilled inthe art can prepare using standard methods, moreover all opticalantipodes, diastereomers and stereomeric forms.

Physiologically tolerable salts of compounds of the formula (I) areunderstood as meaning both their organic and inorganic salts, asdescribed in Remington's Pharmaceutical Sciences (17th edition, page1418 (1985)). On account of the physical and chemical stability and thesolubility, for acidic groups, inter alia, sodium, potassium, calciumand ammonium salts are preferred; for basic groups, inter alia, salts ofhydrochloric acid, sulfuric acid, phosphoric acid or of carboxylic acidsor sulfonic acids, such as, for example, acetic acid, citric acid,benzoic acid, maleic acid, fumaric acid, tartaric acid andp-toluenesulfonic acid are preferred.

Esters, ethers, amides and acetals can be prepared by methods describedin the literature, e.g. in Advanced Organic Synthesis, 4th Edition, J.March, John Wiley & Sons, 1992 or Protective Groups in Organic Synthesis3rd Edition, T. W. Green & P. G. M. Wuts, John Wiley & Sons, 1999.

The carboxyl group can be reduced to the alcohol, for example, usingLiAlH₄ or esterified with the addition of catalytic amounts of aninorganic acid (for example H₂SO₄ or HCl). The hydroxyl groups can beetherified, for example, under the conditions of the Williamson ethersynthesis.

For the detection of the inhibitors of PAI-1, a test is run in which theactivation of a specific substrate by tPA is measured in the presence ofa defined amount of PAI-1 and the substance in each case to beinvestigated. tPA is inhibited by PAI-1. An inhibition of PAI-1 resultsin an increased tPA activity. The enzymatic activity of tPA is measuredcolorimetrically by employing a chromogenic substrate which becomescolored after amidolysis.

IC₅₀ values for the spirobenzofuran lactam derivatives are indicated intable 1:

IC₅₀ Compound of the formula (II) 41 μM Compound of the formula (III) 66μM Compound of the formula (IV) 35 μM

As inhibitors of PAI-1, the compounds according to the invention can beused for the treatment and/or prophylaxis of the diseases mentioned byway of introduction.

The invention therefore further relates to the use of a compound of theformula (I) according to the invention or of a physiologically tolerablesalt thereof as a pharmaceutical in human or veterinary medicine or forthe production of a pharmaceutical in human or veterinary medicine, inparticular for the treatment and/or prophylaxis of coronary heartdisease, acute myocardial infarct, unstable angina pectoris, venousthrombosis and venous thromboembolisms, arterial thrombosis,arteriosclerosis, insulin resistance and macrovascular injuries in typeII diabetes mellitus patients, hypoxia, septic shock, pneumonia andpulmonary fibrosis, and cancers, in particular breast cancer, intestinalcancer, gastric cancer, hepatic cancer, brain tumors, ovarian tumors,esophageal cancer, renal cancer, muscle cell carcinoma, in particularhead and neck muscle carcinoma, particularly preferably a pharmaceuticalfor the inhibition of clotting for the treatment of and/or as aprophylaxis for thromboembolic diseases.

In addition, the present invention relates to a pharmaceuticalcontaining at least one compound of the formula (I), it being possiblein principle for the compound or the compounds of the formula (I) to beadministered as such alone or preferably as a mixture with one or moreof the customary pharmacologically suitable vehicles or excipients.

The compounds according to the invention are stable in the solid stateand in solutions in the pH range between 3 and 8, in particular 5 and 7,and can thus be incorporated into customary pharmaceutical preparations.

The pharmaceuticals according to the invention are in generaladministered orally or parenterally, but rectal administration is alsopossible in principle. Suitable solid or liquid pharmaceuticalpreparations forms are, for example, granules, powders, tablets, coatedtablets, (micro)capsules, suppositories, syrups, emulsions, suspensions,aerosols, drops or injectable solutions in ampule form and preparationshaving a protracted release of active compound, in whose preparationvehicles and additives and/or excipients such as disintegrants, binders,coating agents, swelling agents, glidants or lubricants, flavorings,sweeteners or solubilizers are used.

Customary pharmacologically suitable vehicles or excipients are, forexample, magnesium carbonate, titanium dioxide, lactose, mannitol andother sugars, talc, milk protein, gelatin, starch, vitamins, celluloseand its derivatives, animal or vegetable oils, polyethylene glycols andsolvents, such as, for example, sterile water, alcohols, glycerol andpolyhydric alcohols.

Optionally, the dose units for oral administration can bemicroencapsulated in order to delay the release or to extend it over alonger period of time, such as, for example, by coating or embedding theactive compound in particle form in suitable polymers, waxes or thelike.

Preferably, the pharmaceutical preparations are prepared andadministered in the dose units, each unit containing as activeconstituent a specific dose of one or more compounds of thespirobenzofuran lactam derivatives according to the invention. In thecase of solid dose units such as tablets, capsules and suppositories,this dose can be up to approximately 500 mg, but preferablyapproximately 0.1 to 200 mg, and in the case of injection solutions inampule form up to approximately 200 mg, but preferably approximately 0.5to 100 mg, per day.

The daily dose to be administered is dependent on the body weight, age,gender and condition of the mammal. Under certain circumstances,however, higher or lower daily doses may also be appropriate. Theadministration of the daily dose can be carried out both by singleadministration in the form of an individual dose unit or else in anumber of smaller dose units and by multiple administration ofsubdivided doses at specific intervals.

The pharmaceuticals according to the invention are prepared by bringingone or more of the compounds of the formula (I) according to theinvention, optionally with one or more of the customary vehicles orexcipients, into a suitable administration form.

The invention is illustrated further in the following examples.Percentages relate to the weight. Mixing ratios in the case of liquidsrelate to the volume, unless stated otherwise.

EXAMPLE 1 Preparation of a Glycerol Culture of Stachybotris atraST002348, DSM 14952

100 ml of nutrient solution (malt extract 2.0%, yeast extract 0.2%,glucose 1.0%, (NH₄)₂HPO₄ 0.05%, pH 6.0) were inoculated into a sterile300 ml Erlenmeyer flask with the strain Stachybotris atra ST002348, DSM14952 and incubated on a rotating shaker for 6 days at 25° C. and 140rpm. 1.5 ml of this culture were then diluted with 2.5 ml of 50%strength glycerol and stored at −135° C.

EXAMPLE 2 Preparation of a Preculture in an Erlenmeyer Flask ofStachybotris atra ST002348, DSM 14952

A 300 ml Erlenmeyer flask with 100 ml of the following nutrientsolution: malt extract 2.0%, yeast extract 0.2%, glucose 1.0%,(NH₄)₂HPO₄ 0.05%, pH 6.0, was inoculated with a culture grown on a slanttube/petri dish (same nutrient solution, but with 2% agar) or with 1 mlof a glycerol culture (see example 1) and incubated on a shaker at 140rpm and 25° C.

EXAMPLE 3 Preparation of a Main Culture in an Erlenmeyer Flask ofStachybotris atra ST002348, DSM 14952

A 300 ml Erlenmeyer flask with 100 ml of the following nutrientsolution: 0.5% soluble starch, 0.5% cornstarch, 1% glucose, 0.5% yeastextract, 0.5% “cornsteep” and 0.2% CaCO₃, was inoculated with 2 ml of apreculture (see example 2) and incubated on a shaker at 140 rpm and 25°C. The maximum production of one or more compounds of thespirobenzofuran lactam derivatives according to the invention isachieved after about 120 hours. For the inoculation of 10 l fermenters,a 48 to 96 hour-old submerse culture (inoculation amount about 10%) ofthe same nutrient solution sufficed.

EXAMPLE 4 Preparation of the Spirobenzofuran Lactam Derivatives

A 30 l fermenter was operated under the following conditions:

nutrient medium: 5 g/l of starch 5 g/l of cornstarch 5 g/l of cornsteep,liquid 5 g/l of yeast extract, 5 g/l of CaCO₃ pH 6.0 (beforesterilization) incubation time: 96 hours incubation temperature: 25° C.stirrer speed: 150 rpm aeration: 15 l min⁻¹

By repeated addition of ethanolic polymer solution, it was possible tosuppress foam formation. The production maximum was achieved after about72 to 120 hours.

EXAMPLE 5 Isolation of the Spirobenzofuran Lactam Derivatives from theShaker Cultures of Stachybotris atra ST002348, DSM 14952

After completion of the fermentation of Stachybotris atra ST002348, DSM14952, the culture broth of 50 shaker flasks (in each case 100 ml ofculture broth) was lyophilized together with the biomass and thelyophilizate was extracted with 2×5 liters of methanol. The activecompound-containing, methanolic solution was freed from the residue byfiltration and concentrated in vacuo. The concentrate was diluted withwater and applied to a prepared 1.0 liter MCI GEL, CHP 20P column. Itwas eluted using a gradient of water to 100% acetonitrile in 60 minutes.The column eluate (30 ml per minute) was collected in fractions (30 mleach) and the fractions containing spirobenzofuran lactam derivatives(fr. 26–40) were combined. After concentration in vacuo and subsequentlyophilization, 2.9 g of a pale pink powder were isolated.

EXAMPLE 6 Preliminary Separation of the Spirobenzofuran LactamDerivatives by RP 18 Chromatography

About 1 g of the product obtained according to example 5 was applied toa LiChrospher® 100 RP-18 (e) column (size: 50 mm×250 mm; Merck,Darmstadt) and eluted using a gradient of 20% acetonitrile (+water withaddition of 0.1% trifluoroacetic acid) to 90% acetonitrile with a flowrate of 45 ml per minute. The column eluate was collected in fractions(45 ml), spirobenzofuran lactam derivatives mainly being found infractions 95 to 112. They were combined, freed from the solvent in vacuoand then lyophilized. The yield was 170 mg.

EXAMPLE 7 Purification of the Spirobenzofuran Lactam Derivatives

100 mg of the mixture isolated and concentrated according to example 6were applied to a LUNA® 5μ C18(2) column (size: 21 mm×250 mm) andchromatographed using a gradient of 30 to 85% acetonitrile (+water withaddition of 0.05% TFA). The flow of the eluant was 25 ml per minute, thefraction size 25 ml. Fraction 32 contained the compound of the formula(II) and afforded 6.1 mg after lyophilization. Fraction 33 contained thecompound of the formula (III) and afforded 5.5 mg after lyophilization.Fraction 34 contained the compound (IV) and afforded 5.2 mg afterlyophilization.

The physicochemical and spectroscopic properties of the substancesaccording to the invention be summarized as follows:

EXAMPLE 8 Characterization of the Compound of the Formula (II)

Empirical formula: C₃₂H₃₉NO₆ Molecular weight: 533.67 UV maxima: 248,348

TABLE 2 ¹H and ¹³C chemical shifts of compound (II) in DMSO-d₆ at 300 K(II)

¹H ¹³C^(a))  1 1.73/0.92 23.8  2 1.81/1.40 24.8  3 3.18 73.3  5 2.0139.3  6 1.46/1.40 20.4  7 1.53/1.39 30.7  8 1.77 36.4 11 3.10/2.73 31.612 0.59 15.5 13 0.88 28.6 14 0.79 22.3 15 0.94 15.8 2′-OH 9.68 —  3′6.49 100.8  8′ 4.26 44.3  2″ 5.11 54.5  3″ 3.33/3.27 34.4  5″ 7.22 128.2 6″ 7.25 128.4  7″ 7.14 126.4 ^(a))The values for the ¹³C chemicalshifts are only given to one decimal place, since they were determinedfrom the HMQC spectrum.

EXAMPLE 9 Characterization of the Compound of the Formula (III)

Empirical formula: C₂₉H₄₁NO₆ Molecular weight: 499.65 UV maxima: 248,348

TABLE 3 ¹H and ¹³C chemical shifts of compound (III) in DMSO-d₆ at 300 K(III)

¹H ¹³C^(a))  1 1.74/0.92 23.8  2 1.79/1.39 24.8  3 3.18 73.4  3-OH 4.07—  4 — 37.3  5 2.03 39.4  6 1.44 20.4  7 1.52/1.41 30.7  8 1.79 36.5  9— 98.0 10 — 41.8 11 3.12/2.77 31.6 12 0.66 15.5 13 0.88 28.6 14 0.8022.3 15 0.95 15.7  1′ — 117.0  2′ — 153.8  2′-OH 9.73 —  3′ 6.58 100.9 4′ — 132.8  5′ — 112.2  6′ — 155.8  7′ — 167.9  8′ 4.39/4.28 44.2  1″ —172.1  2″ 4.55 58.3  3″ 2.12 33.8  4″ 0.96 15.7  5″ 1.31/1.05 25.1  6″0.84 10.3 ^(a))The values for the ¹³C chemical shifts are only given toone decimal place, since they were determined from the 2D spectra.

EXAMPLE 10 Characterization of the Compound of the Formula (IV)

Empirical formula: C₂₉H₄₁NO₆ Molecular weight: 499.65 UV maxima: 248,348

TABLE 4 ¹H and ¹³C chemical shifts of compound (IV) in DMSO-d₆ at 300 K(IV)

¹H ¹³C  1 1.75/0.94 23.80  2 1.80/1.41 24.77  3 3.18 73.38  3-OH 4.09 — 4 — 37.22  5 2.03 39.32  6 1.41 20.37  7 1.52/1.41 30.65  8 1.79 36.47 9 — 97.96 10 — 41.72 11 3.13/2.77 31.63 12 0.65 15.48 13 0.88 28.53 140.79 22.29 15 0.95 15.75  1′ — 116.85  2′ — 153.68  2′-OH 9.72 —  3′6.58 100.83  4′ — 133.12  5′ — 112.22  6′ — 155.85  7′ — 168.03  8′4.30/4.25 43.77  1″ — 173.03  2″ 4.81 51.50  3″ 1.98/1.70 37.41  4″ 1.4024.50  5″ 0.91 22.82  6″ 0.87 20.78

EXAMPLE 11 Bioassay for PAI-1 Inhibitors

Reaction: The inhibition of the enzymatic activity of tPA by PAI-1 ismeasured by means of the amidolysis of the chromogenic substrateH-D-IIe-Pro-Arg-pNA (Chromogenix; pNA=para-nitroaniline) as the opticaldensity (OD) at a wavelength of 405 nm.

Test Substances:

Extracts or pure substances, for example of the spirobenzofuran lactamderivatives prepared or characterized in examples 4–10, which arepresent dissolved in DMSO, were diluted in a suitable manner using TRISbuffer pH 8.4.

Method:

5 μl of test substance and 5 μl of PAI-1 are preincubated at roomtemperature for 30 minutes. 10 μl of tPA solution and 20 μl of substratesolution are then added.

The final concentrations in the sample are 50 μM test substance, 4.5 nMPAI-1, 7.5 nM tPA and 1 mM substrate in TRIS buffer pH 8.4. Immediatelyafter the addition of the substrate, the initial absorption is measuredat 405 nm. After incubation at 37° C. for 60 minutes, the absorption ismeasured again.

In each case, blank samples (buffer instead of tPA), positive controls=B(buffer instead of test substances) and tPA controls=A (buffer insteadof PAI-1) are co-tested.

After correction by means of the blank samples, the inhibition isdetermined according to the following equation:

${\%\mspace{14mu}{inhibition}} = {100 - {\frac{{\Delta\;{OD}_{405{nm}}\mspace{14mu}{mean}\mspace{14mu}{value}\mspace{14mu} A} - {\Delta\;{OD}_{405{nm}}\mspace{14mu}{sample}}}{{\Delta\;{OD}_{405{nm}}\mspace{14mu}{mean}\mspace{14mu}{value}\mspace{14mu} A} - {\Delta\;{OD}_{405{nm}}\mspace{14mu}{mean}\mspace{14mu}{value}\mspace{14mu} B}} \times 100}}$

The results of the assay are shown as IC₅₀ values in Table 1.

1. A compound of the formula (I)

wherein R¹ and R² are H, R³ is —OR¹ and R⁴ is benzyl, 2-butyl, or1-(2-methyl)-propyl, or a physiologically tolerable salt of the compoundof the formula (I).
 2. The compound according to claim 1 having thefollowing formula (II)


3. The compound according to claim 1 having the following formula (III)


4. The compound according to claim 1 having the following formula (IV)


5. A process for the preparation of a compound of the formula (I) asclaimed in claim 1, which comprises fermenting the strain Stachybotrisatra ST002348, DSM 14952, or one of its variants or mutants undersuitable conditions in a culture medium until one or more of thecompounds of the formula (I) accumulate in the culture medium and thenisolating it from the culture medium and optionally converting it into aphysiologically tolerable salt.
 6. A pharmaceutical compositioncomprising a pharmaceutically effective amount of at least one compoundof the formula (I) or of a physiologically tolerable salt thereof asclaimed in claim 1, alone or as a mixture with one or more of thecustomary pharmacologically suitable vehicles or excipients.